Protective effects of a triple‐fermented barley extract (FBe) against HCl/EtOH‐induced gastric mucosa damage in mice

Abstract This study was designed to observe the possible protective effects of a triple‐fermented barley (Hordeum vulgare L.) extract (FBe) obtained by saccharification and using Saccharomyces cerevisiae and Weissella cibaria in alleviating gastric damage induced by a hydrochloric acid (HCl) and ethanol (EtOH) mixture in mice. After oral administration of FBe (300, 200, and 100 mg/kg) followed by 1 hr before and after the single treatment of HCl/EtOH (H/E) mixture, the hemorrhagic lesion scores, histopathology of the stomach, gastric nitrate/nitrite content, lipid peroxidation, and antioxidant defense systems including catalase and superoxide dismutase activities were observed. Following a single oral treatment of H/E‐induced gastric damages as measured by hemorrhagic gross lesions and histopathological gastric, ulcerative lesions were significantly and dose‐dependently (p < 0.01 or p < 0.05) inhibited in mice, when all three different doses of FBe were administered as compared to those in H/E control mice. In particular, FBe also increased gastric nitrate/nitrite content and strengthened the antioxidant defense, with a decrease in the level of gastric lipid peroxidation, but increased the activities of CAT and SOD. Moreover, the effects of FBe are comparable to that of ranitidine, a reference drug. The obtained results suggest that this fermented barley extract prevented mice from H/E‐induced gastric mucosal damages through the suppression of inflammatory responses and oxidative stress‐responsive free radicals. Thus, FBe can be useful to treat patients suffering from gastric mucosal disorders as a potent food supplement, and thereby, it would increase the necessity of application in the food industry.

Use of EtOH in in vivo study for inducing gastric ulcer has been commonly used model systems to study on ulcerative pathogenesis and its curative measures (Szabo & Brown, 1987). In addition, to evoke severe and rapid gastric damage induced by EtOH, hydrochloric acid (HCl), which also involved in the progression and pathogenesis of gastric damages, has been additionally administered (Alqasoumi et al., 2009;Huh et al., 2003;Lee et al., 2010;Oyagi et al., 2010). Therefore, administration of the HCl/EtOH mixture has been used as a valuable and simple method to study both the pathogenesis of and the therapy for human ulcerative disease in mouse gastric ulcer models, especially to study natural products based on their potent antioxidant effects (Murata et al., 2012;Niero et al., 2012;Oyagi et al., 2010;Yang et al., 2012).
Some synthetic antiulcer drugs are reported to be used for commercial purposes such as misopostol and Ranitidine (RA), etc.

| Animals and husbandry
The mice (ICR; N = 60), healthy male, mean 38.27 ± 1.81 g, range 25.60-41.80 g, were purchased from OrientBio company (Seungnam, Korea) and used after 10 days of acclimatization. The animals were housed in a polycarbonate cage with maintaining the temperature 20-25°C, humidity 30%-35%, 12-hr light:dark cycle, and ad libitum of water and food. Eight mice for each of the six groups, 48 mice in total, were selected based on body weight (mean 38.27 ± 1.81 g, range 25.60-41.80 g) measured at 1 day before the test material administration after 10 days of acclimatization and were used for further experiments (Table 1 and

| Preparation and administration of test articles
The FBe was prepared and supplied by Glucan Corp. (Busan, Korea) according to methods used in our previous studies Lim et al., 2018a,b and RA were dissolved in distilled water, and orally administered at a volume of 10 ml/kg and 100 mg/kg, following reports of Lim TA B L E 1 Gastric lipid peroxidation, nitrate/nitrite contents, and catalase and SOD activities in the intact or HE-treated mice  (2018a,b) and (Grover et al., 2001). The distilled water at 10 ml/ kg was orally administered as negative control, as demonstrated in Table 1 and Figure 1. All chemicals were purchased from Sigma-Aldrich, St. Louise, MO, USA, company unless otherwise stated. The RA was used as a potent reference drug, according to previous study (Grover et al., 2001).

| Induction of gastric mucosa damage by the HCl/EtOH mixture in mice
One hour after treatment of the vehicle, three different doses of FBe, or RA 100 mg/kg to 24-h fasted mice, an HCl/EtOH mixture (98% EtOH containing 150 mM HCl) was orally administered in a single volume of 5 ml/kg as reported previously (Oyagi et al., 2010).
In intact vehicle control mice, sterilized distilled water was administered as a single gastric gavage, instead of the HCl/EtOH mixture. Both high-grade HCl and EtOH were purchased from Merck (Darmstadt, Germany) and used after adjusting dilutions using distilled water (Table 1 and Figure 1).

| Quantification of gross lesions
After sacrifice, the abdomen was opened with a median incision and the stomach was excised. The excised stomach was opened out along with greater curvature and fixed in 10% formalin so- (2010) and Oyagi Oyagi et al. (2010). For this purpose, the total area of the ulcerous stomach regions was calculated as mm 2 of gastric mucosa.

| Determination of lipid peroxidation or malondialdehyde (MDA) formation
The level of lipid peroxidation in the gastric mucosa was determined by estimating the MDA content following the thiobarbituric acid test (Ohkawa, Ohishi, & Yagi, 1979

| Tissue CAT activity
CAT activity was determined by the method of Evans and Diplock (1991). A homogenate of mouse gastric mucosa was prepared, and the absorbance was measured at 240 nm for 100 s using a UV/ Vis spectrometer. The results were expressed as mM min −1 mg tissue −1 .

| Tissue SOD activity
Gastric SOD activity was determined by a modified method of Minami and Yoshikawa (1979). Briefly, gastric homogenate was used to react with triscacodylate buffer (pH 8.2, 16% Triton X-100, and nitroblue tetrazolium), incubated for 5 min at 37°C, and then measured at 540 nm using a spectrophotometer that expressed as mM min −1 mg wet tissue −1 .

| Gastric nitrate/nitrite contents
Gastric nitric oxide levels were measured as total nitrate/nitrite levels with the use of the Griess reagent (Green et al., 1982). Briefly, the stomach homogenate was mixed with Griess reagent and determined at wavelength of 540 nm using a microplate reader (Tecan, Männedorf, Switzerland). The resulting value was expressed as μM nitrate/nitrite per g of protein, where the protein concentration was detected using a Bradford assay method (Bradford, 1976).

| Histopathology
Approximate regions of the stomach (between cardiac and pylorus, the fundus) were sampled and cross-trimmed across the lumen. All as described by Ku et al. (2009). In addition, lesion invasive percentages in the fundus (%) were also calculated as follows in Equation (1) according to the method of Ku et al. (2009). Semiquantative scoring into four categories; 0 = normal intact mucosa, 1 = slight surface erosive damage, 2 = moderate mucosa damage, and 3 = severe total mucosa damage was based on general and histomorphometric analysis, aforementioned in this experiment.

| Statistical analyses
All numerical data were expressed as mean ± SD of eight determinations. The obtained data were analyzed by one-way ANOVA test followed by least-significant differences (LSD) multicomparison test and Mann-Whitney U (MW) test (Ludbrook, 1997), using SPSS for Windows (Release 14.0K, SPSS Inc., Chicago, IL, USA). Differences were considered significant at p < 0.01 or p < 0.05. In addition, the percent-point changes between intact and H/E control mice were calculated to observe the effects of severities of gastric mucosa damage including ulcerative lesions and the percent-point changes as compared with H/E control and FBe or RA-treated mice were also calculated to evaluate the effects of test substances as described by Equations (2) and (3), and our previous established method (Kang et al., 2014), respectively.

| Effects on lipid peroxidation
The levels of lipid peroxidation and MDA contents in the gastric of H/E-treated control mice were significantly (p < 0.01) increased, compared to those in intact vehicle control mice. However, this increase in MDA content was significantly (p < 0.01) and dosedependently decreased in mice observed with all three doses of FBe. In addition, gastric lipid peroxidation induced by the HCl/EtOH mixture was also significantly (p < 0.01) inhibited after a single oral administration of RA 100 mg/kg, similar to that induced by FBe 200 mg/kg (Table 1).

| Effects on SOD activity
The level of SOD activity in the gastric of H/E control mice was significantly (p < 0.01) decreased, while comparing to those in mice with intact vehicle control. However, this decrease in SOD activity was significantly (p < 0.01 or p < 0.05) and dose-dependently inhibited in mice received different doses of FBe (300, 200, and 100 mg/ kg). In addition, decreases in gastric SOD activities induced by the HCl/EtOH mixture were also significantly (p < 0.01) inhibited after a single oral administration of RA 100 mg/kg, similar to those induced by FBe 200 mg/kg (Table 1).   Figure 4).

| D ISCUSS I ON
Excess EtOH leads to erosion, ulcerative lesions, and petechial bleeding in the mucosa of the stomach and it is rapidly absorbed through gastric mucosa (Eastwood & Kirchner, 1974;Oyagi et al., 2010;Tarnawski et al., 1981). Excess ethanol also generates harmful ROS, superoxide anion, and hydroperoxy free radicals, involved in the pathogenesis of gastric mucosa (Dragland, Senoo, Wake, Holte,  & Blomhoff, 2003;Huh et al., 2003;Oyagi et al., 2010). Gastric mucosa damage can be easily produced by the generation of toxic free radicals (Biswas et al., 2003;Naito et al., 1995) and disorders or decreases in gastric mucosa antioxidant defense systems have been involved in the pathogenesis and progression of gastric ulcers (Kwiecień et al., 2002). EtOH is often used to induce gastric ulcer in various model systems to study on the development of drug for human ulcerative disease (Szabo & Brown, 1987) and to evoke severe and rapid gastric damage together with administration of HCl (Alqasoumi et al., 2009;Huh et al., 2003;Lee et al., 2010;Oyagi et al., 2010). Various synthetic antiulcer drugs are currently available, and some of these including RA, are representative histamine H 2 receptor antagonists specifically used to cure gastric ulcers.
Various fermentation processes increase the bioavailability of phenolic compounds of barley and increase various antioxidantbased pharmacological activities (Giriwono et al., 2010(Giriwono et al., , 2011Hokazono, Omori, Yamamoto, et al., 2010;Iguchi et al., 2009;Kim et al., 2007). In our previous reports on the laxative effects of FBe on normal rats (Lim et al., 2018a) and loperamide-induced constipated rats (Lim et al., 2018b) (Grover et al., 2001) in this experiment. The doses of FBe, 100, 200, and 300 mg/kg, were selected, similar to those in our previous reports on the laxative effects of FBe on normal rats (Lim et al., 2018a) and loperamide-induced constipated rats (Lim et al., 2018b), and the dose level of RA was also selected as 100 mg/kg according to a previous efficacy study (Grover et al., 2001).
The decrease or inhibition of gross hemorrhagic lesion areas is regarded as a valuable indication that test substances have favorable gastric mucosa protective effects based on previous efficacy studies conducted by other researchers (Mori, Hayashi, Iwashima, Matsunaga, & Saito, 2006;Süleyman et al., 2009). Reduced gross lesions indicate more favorable protective effects (Oyagi et al., 2010).

| CON CLUS ION
By assessing the key parameters for protective effects against HCl/

ACK N OWLED G M ENT
Not applicable.

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest.